The present invention refers to recombinant defective adenoviruses containing a DNA sequence coding for hIL-6 (human Interleukin 6) antagonists and/or superantagonists. Such recombinant adenoviruses are used for the preparation of pharmaceutical compositions, in particular in gene therapy for the treatment and/or prevention of diseases caused by hIL-6 overproduction.
Altered hIL-6 serum levels have been described in several pathologies, such as multiple myeloma (1), Castleman""s disease (2), mesangial glomerulonephritis (3), osteoporosis (4), EBV positive lymphomas (5), rheumatoid arthritis (6) and systemic lupus erythematosus (7). In multiple myeloma, IL-6 clearly plays an autocrine and paracrine role in the growth of malignant cells.
It is known from WO 95/26409 and WO 95/25508, in the name of Rhone-Poulenc Rorer S.A., the use of recombinant defective adenoviruses to achieve stable expression of a protein of interest (in particular a cytokine as in the case of WO 95/26409), whose coding sequence is inserted in the defective adenovirus genome under the control of a strong promoter. It is also known that, in the case of adenovirus mediated gene therapy, inflammation represents a major obstacle, being the cause for tissue injury as well as, at least in part, for termination of gene expression (9) (10). There are indications that IL-6 is the major inflammatory cytokine produced after adenovirus injection (11) (12).
Now, it has been unexpectedly found that mutants of hIL-6 with antagonist or superantagonist activity over hIL-6 (disclosed in WO/00852 in the name of the present Applicant) produced by cells infected both in vitro and in vivo with a recombinant defective adenovirus, containing a DNA sequence coding for said antagonists or superantagonists under the control of a strong promoter, inhibit hIL-6 activity on a variety of human cells. Therefore, said recombinant adenoviruses can be used to prolong adenovirus mediated gene of interest expression in vivo.
The subject matter of the present invention is a recombinant defective adenovirus comprising a DNA or a cDNA sequence coding for a mutant of human Interleukin 6, a fragment thereof or a derivative thereof, with antagonist or superantagonist activity over hIL-6.
In preferred embodiments of the invention, the hIL-6 mutants with superantagonist activity over hIL-6 are Tyr31Asp, Gly35Phe, Ser118Arg, Val121Asp, Gln175Ile, Ser176Arg, Gln183Ala and Tyr31Asp, Gly35Phe, Leu57Asp, Glu59Phe, Asn60Trp, Gln75Tyr, Ser76Lys, Ser118Arg, Val121Asp, Gln175Ile, Ser176Arg, Gln183Ala (hereinafter referred to also as Sant1 and Sant7 respectively).
The recombinant defective adenovirus of the invention can be put under the control of signals allowing its expression in different kinds of human specific cells. The expression signals can be selected among the viral promoters. The viral promoter can be Rous Sarcoma Virus (RSV).
The invention also refers to the use of the recombinant defective adenovirus, comprising at least a DNA or a cDNA sequence coding for a mutant of hIL-6, a fragment thereof or a derivative thereof, with antagonist or superantagonist activity over hIL-6, for treating and/or preventing diseases such as multiple myeloma, Castleman""s disease, mesangial glomerulonephritis, osteoporosis, EBV positive lymphomas, rheumatoid arthritis and systemic lupus erythematosus.
Pharmaceutical compositions comprising at least one of the above recombinant defective adenoviruses are also the subject matter of the present invention. The pharmaceutical compositions can be in injectable form. They can contain the recombinant defective adenovirus in the range from 104 to 1014 pfu/ml (plaque forming unit/ml), preferably from 106 to 1010 pfu/ml. Mammalian cells, in particular human cells, infected by at least one of the above recombinant adenoviruses are a further subject of the present invention. These cells can additionally express a gene of interest coding for a therapeutic protein. These cells can be further infected with at least one virus expressing a therapeutic gene.
The infected human cells can be selected from the group comprising fibroblasts, myoblasts, hepatocytes, endothelial cells, glial cells and keratinocytes.
Another subject matter of the present invention is an implant comprising the cells infected by at least one of the above recombinant defective adenoviruses and an extracellular matrix. The extracellular matrix can be a gelifying compound which can be selected from the group consisting of collagen, gelatin, glucosaminoglycans, fibronectins and lectins. The extracellular matrix can comprise a support for anchoring the infected cells. The support can be preferably made of polytetrafluoroethylene fibers.
The objects, characteristics and advantages of the invention will be further illustrated, although not limited, by the following examples directed to embodiments thereof.